Advanced cancers represent one of the major causes of human death, but no fully effective methods of treatment have been suggested so far. Cell-based immune therapies represent the most promising non-toxic method of cancer treatment. Cancer immunotherapy aims to destroy tumor cells by immunological mechanisms. It may be used as a sole treatment, or as an adjuvant for other types of therapies such as e.g. surgery, irradiation and chemotherapy. The strategy is based on ex vivo manipulation and reintroduction of cellular products to circumvent immune competences for the purpose of inducing tumor specific immune responses. Thus, the ultimate goal of such cell-based immune therapies is the induction of tumor-specific effector cells and recent advances has focused on CD8+ cytotoxic T lymphocytes (CTL) capable of recognizing and killing tumor cells. It is important that these CTL should be able to survive for a long time in the organism in form of memory cells that can rapidly be reactivated and expanded upon re-appearance of tumor cells in the organism.
The possibility to employ the immune system to attack cancer cells follows from the fact that cancer cells contain new proteins or over-express the existing proteins that may become targets for the immunological attack. Significant number of tumor-associated antigens recognized by such CTLs has been identified since description of the first human tumor antigen MAGE-1 in 1991 by T. Boon's group (Novellino et al., 2005). Three major groups of tumor antigens are currently under intensive investigation as possible targets for active immunotherapy: (i) differentiation antigens such as MART-1, gp100 and tyrosinase for melanomas, and PSA and PSMA for prostate cancer; (ii) overexpressed antigens such as telomerase and survivin and (iii) cancer/testis antigens (CTA) such as MAGE, GAGE, NY-ESO-1 and BORIS.
From the point of specific targeting of immune response to tumor cells, the most perspective are CTA, as they are not expressed in normal cells except germ cells of testis which are not recognized by the immune system (germ cells lack expression of histocompatibility molecules) (see reviews on CTA: (Kirkin et al., 2002; Zendman et al., 2003)). It was demonstrated that activation of CTA in tumor cells is due to promoter demethylation at CpG regions and is a consequence of genome-wide demethylation process that occurs in many cancers, and is correlated with tumor progression (De Smet et al., 1996). Indeed, CTA expression was shown to be associated with tumor progression (Brasseur et al., 1995; Eura et al., 1995; Katano et al., 1997; Patard et al., 1995). Therapeutic potentials of this group of tumor antigens have been confirmed by a number of studies. For example, (a) melanoma patient MZ-2 with metastases was subjected to multiple vaccinations with killed autologous tumor cells leading to development of CTL responses against several CTA (MAGE-1, MAGE-3, GAGE and BAGE, see review (van Pel et al., 1995)) experienced long-lasting disease-free period (P. Coulie, personal communication); (b) vaccination of melanoma patients with dendritic cells loaded with peptide from MAGE-A3 induced regression of antigen-positive metastases (Thurner et al., 1999a); and (c) therapeutic efficiency of polyvalent vaccine correlated with induction of immune response against MAGE-3 antigen (Reynolds et al., 2003). Nevertheless, due to high heterogeneity in the expression of separate CTA members, this group of antigens is not popular as immunological target compared to differentiation or over-expressed antigens.
In order to overcome the problem with heterogenous expression of CTA, vaccines targeting these antigens should be as polyvalent as possible. The patent application WO 03/045427 describes such a polyvalent vaccine targeting CTA that is based on pre-selected human melanoma cell line that express high levels of CTA and do not express melanocyte differentiation antigens. All other tumor cell-based vaccines either employ autologous tumors, or standard cell lines selected on the principle “the more different tumor antigens, the better”. Employment of autologous tumor cells in a vaccine as a source of antigens may in some cases induce tumor regression, when it is applied in the form of dendritic cell-based vaccination (O'Rourke et al., 2003; Marshall et al., 2006). The major limitation of the polyvalent vaccine approach is difficulty in production and standardisation of the antigenic mixture. Direct use of original biopsy material lacks standardization, and also as well as such a material is often not available. Use of standard cell lines has not shown high clinical efficiency so far (Palucka et al., 2006; Salcedo et al., 2005).
One of the reasons for low efficiency of using whole tumor cell material is low efficiency of cross-presentation. In order to stimulate generation of the cytotoxic T cells, exogenous tumor protein antigens need to be taken up by antigen presenting cells through the process of endocytosis and then transferred from endocytic vesicles into the cytosol (see review (Cresswell et al., 2005)). This is required for the processing of protein antigens and formation of antigenic peptides. Later on the peptides are presented on the surface in association with MHC class I molecules. Only a small portion of exogenously added antigens enters the cell and only a small fraction of the protein taken up thereafter undergoes cross-presentation. In order to overcome the low efficiency of cross-presentation, several authors suggested use of RNA isolated from tumor cells for transfection of antigen presenting cells (Gilboa and Vieweg, 2004; Schaft et al., 2005; Kyte et al., 2006). The major limiting factor of RNA transfection is lack of formation of complexes of antigenic peptides and MHC class II molecules that is required for induction of T-helper response. Without T-helper response no formation of the CD8+ immunological memory is taking place (Bevan, 2004; Castellino and Germain, 2006), and without formation of memory, CD8+ CTLs die after the initial expansion. In addition, under conditions of tumor growth, formation of CD8+ memory response may be corrupted due to the presence of regulatory cells (Klebanoff et al., 2006).
5-Aza-2′-deoxycytidine (5-Aza-CdR) has been known to induce expression of cancer/testis antigens primarily in tumor cells (Weber et al., 1994). Also, it was previously demonstrated by one group (De Smet et al., 1996) but not another (Weber et al., 1994) that 5-Aza-CdR might also induce expression of at least one of CTA, MAGE-A1, in PHA-activated peripheral blood lymphocytes.
This technique is used in the patent application WO 03/012086 as part of a method of generating antigen presenting cells which comprises collecting cells from a subject, activating the cells with agents such as pokeweed mitogen (PWM) and phytohemagglutinin (PHA), culturing the activated cells ex vivo, and treating the cultured cells with DNA hypomethylating agents so that the cells express multiple tumor associated antigens (CTA). The CTA produced in this way are proposed for use as cancer vaccines. However a major issue in this procedure is the use of foreign agents such as pokeweed mitogen (PWM) and phytohemagglutinin (PHA) for activation the cells.
There is still an unmet requirement for development of effective cell-based vaccine that will be able to overcome these problems and induce productive and long-lasting CTL-mediated anti-tumor immune response.
It is the aim of the present invention to provide a process for preparing antigen presenting compositions by a chemical treatment in which only normal cells are used and no foreign agents are used to activate the cells.